Seeing White: Management of TIVA during autologous breast reconstruction.

Total IntraVenous Anaesthesia is frequently the anaesthetic of choice for enhanced recovery after surgery pathways during breast reconstruction free flap surgery. This relies upon the continuous intravenous infusion of propofol. We describe our experience of two patients where augmentation of a venously congested DIEP flap with a cephalic vein transposition procedure, risked interruption of the intravenous delivery of anaesthesia to the patient. We also share our steps taken to mitigate this risk going forward.

Physicochemical and sensory properties of ice-cream formulated with virgin coconut oil.

The substitution of milk fat with virgin coconut oil (VCO) was used to produce nutritious ice cream with pleasant coconut flavor and aroma. Three formulations were developed whereby formulation VCO4, VCO8 and VCO12 was substituted with 4%, 8% and 12% of VCO, respectively. The physicochemical properties of ice creams analyzed include overrun, meltdown, pH, titratable acidity, total solid, protein and fat content. The fatty acids profile of VCO formulated ice creams and their stabilities over 3 and 6 weeks storage were studied respectively using gas chromatography (GC). Qualitative descriptive analysis (QDA) and consumer affective test were performed among the trained and untrained panelists. Significant differences (p < 0.05) of overrun, pH, total solid, protein and fat content between ice cream formulations were observed except titratable acidity. Increased VCO content in ice cream formulations lowered the melting resistance of ice cream. For GC analysis, the major fatty acid identified was lauric acid. Upon storage time, the concentration of unsaturated fatty acid decreased but the concentration of saturated fatty acid increased. The result of QDA showed that formulation VCO4, VCO8 and VCO12 were significantly (p < 0.05) different in attributes of color, firmness and smoothness as compared to the control ice cream. Formulation VCO12 was highly accepted by panelists in terms of the acceptance level of appearance, aroma, texture, flavor and overall acceptability. Hence, it has a potential marketable value.

Bleeding risk and platelet transfusion refractoriness in patients with acute myelogenous leukemia who undergo autologous stem cell transplantation.

Therapy for acute myelogenous leukemia can be complicated by alloimmunization to histocompatibility antigens (HLA), with resultant refractoriness to platelet transfusions. Autologous peripheral blood or bone marrow stem cell transplantation (referred here collectively as 'autoBMT') is emerging as a standard consolidative strategy in acute myelogenous leukemia (AML). We had noted life-threatening bleeding associated with platelet transfusion refractoriness following autoBMT; we therefore retrospectively analyzed 39 AML patients for this complication following BMT. All patients received high-dose chemoradiotherapy, followed by infusion of allogeneic sibling donor (n = 12, alloBMT) or autologous (n = 27, autoBMT) stem cells. HLA alloimmunization was assessed if patients were suspected of immune refractoriness to random donor platelet transfusions. Within 100 days of stem cell infusion, one of three alloBMT and six of 12 autoBMT recipients tested were HLA alloimmunized (not statistically significant, NS). Five of six HLA alloimmunized autoBMT patients experienced delayed bleeding, which contributed to their demise while still in remission (P < 0.001). Increased platelet requirements in HLA alloimmunized autoBMT recipients were observed between days 61 and 100 post-BMT, at a median of 211 platelet transfusions vs 0 in non-alloimmunized autoBMT patients (P < 0.01) and 17 in alloBMT patients. Our data suggest that platelet transfusion refractoriness, when associated with HLA alloimmunization, is a risk factor for increased platelet transfusion requirements, delayed bleeding, and poor outcome following autoBMT for AML.

A novel HLA-A*8001 allele identified in an African-American population.

HLA polymorphism varies among different racial origins. Certain antigens are restricted to a particular ethnic group, suggesting that genetic events might have generated further unique polymorphism following racial diversification. In this study, we have identified and characterized a novel HLA-A allele, officially designated A*8001. The class I molecule, HLA-AXBG, encoded by this allele could not be defined by commonly available HLA antisera. We have identified two alloantisera that appear to be monospecific for this new antigen. The observed frequency of AXBG antigen specificity is 2% in African Americans among the total of 254 tested, but it has not been found in 305 Caucasians tested. IEF and cDNA sequencing analyses on multiple individuals revealed that AXBG antigen defined by the two antisera are encoded by an identical HLA-A*8001 allele and it has a number of unique amino acid residues that have not been observed among other HLA-A alleles but are known to occur in certain human nonclassic class I genes and nonhuman primate class I genes. The ability to identify the new HLA-AXBG antigen will improve the precision of HLA-A typing in black populations and enable us to match a patient with a donor for this otherwise undefined antigen in transplantation.

The role of HLA-DPB1 disparity in the development of acute graft-versus-host disease following unrelated donor marrow transplantation.

The role of HLA-DPB1 disparity in the development of acute graft-versus-host disease (GVHD) following unrelated donor (URD) marrow transplantation is unknown. We studied 129 patients who underwent marrow transplantation from HLA-A, -B, -DRB, and -DQB matched URDs to determine whether matching for HLA-DPB1 alleles significantly decreased the risk of developing acute GVHD. HLA-DPB1 alleles were determined by sequence-specific oligonucleotide hybridization and by the number of patient DPB1 alleles not shared by the donor scored. The Kaplan-Meier probability of developing grades II to IV acute GVHD was determined for patients incompatible for zero (group A), one (group B), or two (group C) DPB1 alleles. Of the 129 pairs, there was no recipient DPB1 incompatibility in 28 (22%), one DPB1 mismatch in 72 (56%), and two DPB1 mismatches in 29 (22%). The probability of grades II to IV acute GVHD was 0.69 (0.50, 0.86) for group A, 0.83 (0.73, 0.91) for group B, and 0.72 (0.56, 0.87) for group C (P = .63). These results indicate that matching patients and unrelated donors for HLA-A, -B, -DRB, and -DQB does not predict for matching at DPB1. However, recipient incompatibility for DPB1 alleles does not detectably influence the risk of acute GVHD. Therefore, HLA-DP disparity should not be used as an exclusion criterion for donor selection in unrelated marrow transplantation.

Selection of an unrelated donor for marrow transplantation facilitated by the molecular characterization of a novel HLA-A allele.

Precise HLA typing is crucial in the selection of marrow donors for the treatment of patients with hematologic malignancy. This study was undertaken to characterize an unusual variant of HLA-A30, designated HLA-A30JS, identified in a patient with leukemia who was a candidate for unrelated donor marrow transplantation. IEF and cDNA-sequencing analyses revealed that A30JS is a novel variant differing from the IEF-defined subtype A30.1 (encoded by the A*3002 allele) by a single amino acid substitution. An unrelated marrow donor was identified who was matched with the patient for HLA-A3, B7, B18, DR2, and DR3, but mismatched within the A30 antigen family for the two distinct alleles A*3002 versus A30JS. These two alleles encode a single amino acid substitution, Arg versus Gly, at position 56 in the alpha 1 domain. Position 56 is located outside the antigen-binding cleft of the class I molecule, suggesting that this substitution may not be functionally significant. Transplantation from this donor was performed and the patient is surviving free of leukemia for more than 700 days after transplant. The maximum acute GVHD observed was scored as grade II, but immunosuppressive therapy is still required for control of chronic GVHD. This study demonstrates how the molecular characterization of a novel HLA-A allele in a patient could facilitate the selection of an unrelated donor. Lacking this information, it would not have been possible to select a donor for this patient, and thus apparently successful marrow transplant could not have occurred.

Marrow transplantation from unrelated donors for treatment of hematologic malignancies: effect of mismatching for one HLA locus.

One hundred twelve patients less than 36 years old received marrow grafts from unrelated donors as treatment for hematologic malignancy. Seventy donor/recipient pairs were phenotypically identical for HLA-A, -B, and -D, while 42 had a "minor" disparity at one HLA locus. There was an increase in the risk of acute graft-versus-host disease (GVHD) in patients receiving HLA-partially matched grafts compared with those receiving HLA-matched grafts (51% v 36% probability of grades III-IV acute GVHD). However, in this cohort of patients, there was no significant difference in survival (at 1.5 years, 46% v 51% for good-risk patients, 44% v 30% for poor-risk patients). This finding suggests that some degree of HLA disparity can be tolerated in young patients transplanted from unrelated donors for malignant disease, thus making transplantation an option available to larger numbers of patients.

Allelic variations clustered in the antigen binding sites of HLA-Bw62 molecules.

HLA-Bw62 is a serologically defined class I antigen specificity, but we show that it represents a family of five distinct alleles in this study. Five variants of HLA-Bw62 antigens were identified by isoelectric focusing, and sequencing studies revealed that these are a family of closely related alleles differing from one another by one to six amino acid substitutions at eight positions: 63 in the alpha 1 domain and 94, 95, 97, 99, 113, 152, and 156 in the alpha 2 domain. These substitutions are located in the two alpha-helices and two adjacent beta-strands, and the side chains of most amino acids face into the antigen binding groove. Functional assays using an in vitro generated Epstein-Barr virus (EBV)-specific Bw62-restricted cytotoxic T lymphocyte clone indicated that the minimal structural variations located in the antigen binding sites of the HLA-Bw62 variant molecules could affect the presentation of the nominal EBV antigen. This study revealed that the HLA-Bw62 antigen family consists of at least five closely related alleles, and further demonstrated that these alleles with minimal structural variations might play distinct functional roles in regard to antigen presentation.

Risk of gastric cancer among Korean familial adenomatous polyposis patients. Report of three cases.

Gastric cancer has been recognized as an extracolonic manifestation in patients with familial adenomatous polyposis (FAP). In Korea, gastric cancer is the most common malignant neoplasm. In a recent survey, we collected data from 72 Korean patients with FAP. Among them, three (4.2 percent) were found to have associated gastric cancer. This incidence of gastric cancer in our series is much higher than the previous reports from Japan and other countries. The expected cumulative incidence of gastric cancer among these 72 patients was 0.44, which gives the standardized incidence ratio of 6.9 (95 percent CI, 1.4-20.1). This difference in incidence of gastric cancer was statistically significant (P less than 0.05), which implies that patients with FAP are at significantly higher risk of developing gastric cancer compared with the general population in Korea. These findings confirm an increased risk of gastric cancer in FAP patients, even in a region where gastric cancer is highly prevalent.

Molecular typing of HLA-B27 alleles.

HLA-B27 represents a family of closely related antigens. Six alleles which differ in a limited number of nucleotide substitutions have been described (B*2701--B*2706). These changes are clustered in alpha 1 and alpha 2 domains. Polymerase chain reaction strategies were designed to amplify specific regions of class I exons 2 and 3. Amplified sequences were tested with eight sequence-specific oligonucleotides to distinguish all B27 subtypes. We also subtyped B27 in 50 healthy Spanish individuals using this procedure. The B*2705 subtype is over-represented in our population (96%). The remaining 4% carried the B*2702 allele. This finding is in agreement with the frequencies described by other techniques (cytotoxic T lymphocytes and isoelectric-focusing) for Caucasian populations. Class I oligotyping is a poorly developed field with significant potential applications. This procedure of genotyping B27 alleles is a reliable method which can be used in transplantation and B27-associated disease studies.

A novel HLA-B27 allele maps B27 allospecificity to the region around position 70 in the alpha 1 domain.

There are six known HLA-B alleles that share the HLA-B27 allospecificity, yet differ by one to six amino acid substitutions. Each of these B27 alleles can be readily assigned by one of the six representative IEF patterns. Two unrelated individuals, LH and HS, express B27 Ag that appear to be identical by IEF, but an HLA-B27 alloreactive CTL clone I-73 was found to react differently with these cells, suggesting these B27 molecules are not identical. We sequenced polymerase chain reaction-amplified B27 cDNA clones obtained from HS and compared its deduced amino acid sequence (B27-HS) with the B27 sequence of LH (B27-LH) which was previously designated the B*2701 allele. B27-HS and B27-LH differ by eight amino acids; three in alpha 1 domain and five in alpha 2 domain. These amino acid substitutions of B27-HS altered T cell recognition but not the B27 serologic epitope or IEF pattern. B27-HS differs from the six known B27 alleles by five to eight amino acid substitutions, and thus it represents the seventh allele of the HLA-B27 Ag family. This novel B27 allele might have been derived from a gene conversion event. Previously, two amino acid residues at positions 70 and 97 were suggested to be specific for B27 Ag family. B27-HS now reveals that Lys at position 70 is specific for B27 but Asn at position 97 is not. We propose that the region around position 70 might be crucial in determining the B27 serologic epitope and possibly in peptide Ag binding. This study also demonstrates that class I molecules of the same Ag specificity sharing an indistinguishable IEF pattern are not necessarily identical, and indicates that only the definitive determination of primary structure would identify all the class I alleles that are functionally relevant in regard to alloreactivity, T cell restriction, and disease association.

Association of four HLA class III region genomic markers with HLA haplotypes.

We have studied restriction fragment length polymorphism (RFLP) in the region 300 kb centromeric to the HLA-B locus. Four probes were used: one was genomic DNA derived from the tumor-necrosis factor (TNF)-beta gene, one was a cDNA for the BAT3 gene, and two single-copy genomic probes, R5A and M20A. The order of these markers from HLA-B towards the centromere is M20A, R5A, TNF and BAT3. The BAT3 and TNF-beta probes each detected two allelic bands with Taq I and Nco I digestion, respectively; the R5A and M20A probes each detected three polymorphic allelic bands with BstEII digestion. To determine if these restriction polymorphisms are preferentially associated with certain HLA-B and -DR haplotypes, a total of 153 HLA haplotypes was analyzed. The haplotypes A1, B8, DR3 and A3, B7, DR2 were each associated with a distinct combination of polymorphisms identified at these four sites, thereby demonstrating that the strong linkage disequilibrium characteristic of these haplotypes extends also to this segment of the class III region. In contrast, haplotypes that are not in positive linkage disequilibrium, such as A1,B8,DR4 and A2,B7,DR3, showed no preferential association with any of these polymorphisms. The antigens HLA-B27 and B35 were also found to be in positive linkage disequilibrium with RFLP patterns at three of these sites, and HLA-B14,B35,B44,Bw57 and Bw62 were found preferentially associated with polymorphisms at one or two of these sites, independent of the DR antigen present. These data further demonstrate that genetic linkage disequilibrium in the HLA class III region is complex and variable among different HLA haplotypes.

Immunogenetics of the HLA system.

The study of the HLA system was primarily initiated to understand the basis for the histocompatibility between recipients and tissue donors. HLA typing methods are being continuously improved and biochemical and molecular typing, in particular, are expected to provide precise typing of the HLA system. Conventional HLA typing methods can define antigen specificities, while biochemical and molecular methods will provide direct allele typing that is based on the actual sequence polymorphism. The precise tissue typing will definitely improve the outcome of transplantation. Structural studies have revealed the highly polymorphic nature of the HLA system and given insight to understanding the molecular basis of the HLA polymorphism. One big immunological puzzle remaining to be answered is how T-cell receptor molecules recognize peptide antigen in conjunction with the HLA molecule. The crystallization of the T-cell receptor molecule, an experiment currently underway, will eventually reveal the structural basis of the trimolecular interaction.

The HLA system in clinical marrow transplantation.

The HLA system is comprised of more than 30 class I and class II genes that encode a polymorphic array of cell-surface glycoprotein molecules that function to restrict or direct the specificity of T-cell responses. Class I alloantigens, encoded by HLA-A, -B, and -C genes, historically have been identified and characterized by the use of alloantisera. Three additional class I genes, HLA-E, -F, and -G, have been identified recently, but it is not known yet if these are relevant to transplantation. The demonstration of further polymorphism among class I alleles, however, has been made possible by the use of cytotoxic lymphocytes and by isoelectric focusing gel electrophoresis. Class II alloantigens, encoded by DR, DQ, and DP genes residing in the HLA-D region, can be defined both serologically and by the use of cellular reagents. Recent advances in DNA typing methods, including restriction fragment length polymorphism and sequence-specific oligonucleotide probe analysis, provide tools which more completely define the extent of HLA polymorphism within given populations. The diversity of allelic variation within the HLA system, coupled with the fundamental role of class I and class II molecules in the triggering of allograft reactions, necessitates the continuing improvement of techniques for characterizing distinct HLA molecules and providing for the better matching of donor and recipient prior to allotransplantation.

Molecular mimicry in HLA-B27-related arthritis.

A unique feature of patients with ankylosing spondylitis and reactive arthritis is that almost all share the HLA type B27. The primary structures of the HLA-B27 antigens have been determined. At least six variants exist. However, disease predisposition does not appear to be restricted to a particular variant. One hypothesis about the pathogenesis of arthritis is that the bacteria that cause the arthritis carry components that are cross-reactive with HLA-B27 antigens. Several reactive bacterial components have indeed been identified using monoclonal anti-HLA-B27 antibodies. Even more striking is the identification, through a computerized search, of a Klebsiella protein. This protein carries a stretch of six amino acids identical to residues 72 to 77 of two of the HLA-B27 variants. A synthetic peptide carrying these six amino acids of HLA-B27 protein is reactive with serum antibodies in some patients with arthritis. With this knowledge, investigators will be able to formulate new approaches for examining the pathogenesis of HLA-B27-associated arthritis.

Molecular analysis of the variant alloantigen HLA-B27d (HLA-B*2703) identifies a unique single amino acid substitution.

HLA-B27 is a human major histocompatibility complex class I product defined by its antigenic specificity with conventional alloantisera. Detailed studies using monoclonal antibodies, cytotoxic T lymphocytes (CTL), and isoelectric focusing (IEF) gel electrophoresis demonstrated the heterogeneity in the B27 antigen. We have previously identified a unique variant molecule of B27 designated locally as B27d which is distinguished from other B27 variants by isoelectric point, serologic reactivity, and by a cloned CTL recognition. A gene encoding the B27d variant has been cloned and a complete nucleotide sequence has been determined. Compared to the sequence of the prototype B27a, the B27d has a single base substitution at codon 59 (B27a:TAT--B27d: CAT) in exon 2 responsible for Tyr to His substitution. A His residue at this position in the alpha 1 domain is unique among the known class I sequences and this single amino acid change is apparently sufficient to alter the epitope(s) recognized by antibody and cytotoxic T cell receptor. Previous primary structural analysis of the other five B27 variants has revealed differences of two to four amino acids. The combined structural data on the B27 variants indicate that (1) HLA-B27 represents a family of closely related B locus alleles that share the B27 allospecificity and differ by a limited number (one to four) of amino acid substitutions and (2) point mutation as well as gene conversion might be the mechanism responsible for the allelic variation of B27 antigen family.

Structural analysis of an HLA-B27 functional variant, B27d, detected in American blacks.

The structure of a new functional variant B27d has been established by comparative peptide mapping and radiochemical sequencing. This analysis completes the structural characterization of the six known histocompatibility leukocyte antigen (HLA)-B27 subtypes. The only detected amino acid change between the main HLA-B27.1 subtype and B27d is that of Tyr59 to His59. Position 59 has not been previously found to vary among class I HLA or H-2 antigens. Such substitution accounts for the reported isoelectric focusing pattern of this variant. HLA-B27d is the only B27 variant found to differ from other subtypes by a single amino acid replacement. The nature of the change is compatible with its origin by a point mutation from HLA-B27.1. Because B27d was found only in American blacks and in no other ethnic groups, it is suggested that this variant originated as a result of a mutation of the B27.1 gene that occurred within the black population.

Structural analysis of an HLA-B27 population variant, B27f. Multiple patterns of amino acid changes within a single polypeptide segment generate polymorphism in HLA-B27.

The structure of a new HLA-B27 variant, B27f, distinguishable from other HLA-B27 subtypes by isoelectric focusing and serologic criteria, has been established by comparative peptide mapping and radiochemical sequence analysis. HLA-B27f differs from the major B27.1 subtype in three clustered amino acid replacements: Asp74, Asp77, and Leu81 in B27.1 are changed to Tyr74, Asn77, and Ala81, respectively in B27f. This pattern of differences is analogous to that of HLA-B27.2 in that this subtype also differs from B27.1 in multiple clustered substitutions within the same segment. Thus, polymorphism within the HLA-B27 system is being achieved by introducing different sets of amino acid changes within a particular short segment of the alpha 1 domain. The most likely mechanism for the introduction of multiple changes within this segment is a nonreciprocal recombination event, such as gene conversion. The structural analogies and ethnic distribution of B27f and B27.2 as compared with those of B27.3, and B27.4 support a dynamic model of HLA-B27 evolution in which polymorphism has been created after the separation of the major ethnic groups. In this model, a Caucasian branch would be characterized by subtypes differing from B27.1 in a few changes within the alpha 1 domain, which were probably generated by single genetic steps. An Oriental branch would include those subtypes which differ from B27.1 by changes in both alpha 1 and alpha 2, involving multiple genetic steps for their generation.